Abstract:

The present invention provides novel in vitro assays for determining the
nephrotoxicity of a compound. These assays correlate well with in vivo
nephrotoxicity and also provide high-throughput methods to screen
multiple compounds for in vivo nephrotoxicity. In addition, the methods
of the present invention may be adapted to screen for nephroprotectant
compounds, including those that protect cells and animals from the
nephrotoxic effects of aminoglycoside antibiotics.

Claims:

1. A method for determining the nephrotoxicity of one or more test
compounds, said method comprising:(i) contacting discrete populations of
HK-2 cells with one or more test compounds;wherein a plurality of
different concentrations for each of said one or more test compounds
contacts a separate discrete population of HK-2 cells;(ii) determining
the level of an indicator of nephrotoxicity for each of said populations
of HK-2 cells contacted in step (i) to produce a dose response curve for
each of the one or more test compounds; and(iii) determining the
nephrotoxicity of each of said one or more test compounds.

2. The method of claim 1, wherein each of the populations of HK-2 cells
are located in separate wells in a tissue culture device comprising a
plurality of wells.

3. The method of claim 1, further comprising:(iv) contacting additional
discrete populations of HK-2 cells with a plurality of concentrations of
one or more control nephrotoxic compounds;(v) determining the level of an
indicator of nephrotoxicity for each of said additionally contacted
populations of HK-2 cells contacted in step (iv) to produce a dose
response curve for each of said one or more control nephrotoxic
compounds; and(vi) determining the nephrotoxicity of each of the one or
more control nephrotoxic compounds.

4. The method of claim 3, wherein the plurality of concentrations of the
one or more test compounds tested and the plurality of concentrations of
the one or more control nephrotoxic compounds tested are in the range of
about 1 mg/mL to about 1 ug/mL.

5. The method of claim 3, wherein the determination of the nephrotoxicity
of each of said one or more compounds comprises calculating an EC50 for
each of the one or more test compounds from the dose response curves of
step (ii).

6. The method of claim 5, wherein the determination of the nephrotoxicity
of each of said one or more compounds comprises comparing the EC50 for
each of the one or more test compounds to an EC50 for each of the one or
more nephrotoxic compounds calculated from the dose response curves from
step (iv).

7. The method of claim 3, wherein the one or more control nephrotoxic
compounds are selected from the group consisting of: amikacin,
gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin,
tobramycin, apramycin.

8. The method of claim 1, wherein the one or more test compounds are
aminoglycosides.

9. The method of claim 8, wherein the indicator of nephrotoxicity is
apoptosis.

10. A method for determining the ability of one or more candidate
nephroprotectant compounds to act as a nephroprotectant, said method
comprising:(i) contacting discrete populations of HK-2 cells with a
plurality of different concentrations of one or more candidate
nephroprotectant compounds in the presence of a nephrotoxic compound,
wherein each of a plurality of different concentrations for each of said
one or more candidate nephroprotectant compounds contacts a separate
discrete population of HK-2 cells;(ii) determining the levels of an
indicator of nephrotoxicity for each one of the contacted populations of
HK-2 cells of step (i); and(iii) determining the ability of each one or
more candidate nephroprotectant compounds to act as a nephroprotectant.

11. The method of claim 10, further comprising:(iv) contacting additional
discrete populations of HK-2 cells with a plurality of different
concentrations of said one or more candidate nephroprotectant compounds
in the absence of a nephrotoxic compound, wherein each of a plurality of
different concentrations for each of said one or more candidate
nephroprotectant compounds contacts a separate discrete population of
HK-2 cells;wherein the ability of a candidate nephroprotectant compound
to act as a nephroprotect is determined when a dose-dependent decrease in
the indicator of nephrotoxicity is present in the contacted HK-2 cells of
step (i) compared to the indicator of nephrotoxicity in the contacted
HK-2 cells of step (iii) for a given candidate nephroprotectant compound.

12. The method of claim 11, wherein the indicator of nephrotoxicity is
apoptosis.

13. The method of claim 12, wherein the indicator of nephrotoxicity is
caspase activity.

14. The method of claim 10, said method further comprising:(iv) validating
the ability of said one or more candidate nephroprotectant compounds to
act as a nephroprotectant by performing one or more counterscreens.

15. The method of claim 14, wherein said one or more counterscreens are
selected from the group consisting of: a cell viability assay, and an
assay for luciferase activity.

16. The method of claim 15, wherein the counterscreens are a cell
viability assay and an assay for luciferase activity.

17. The method of claim 16, wherein the luciferase activity is produced in
the presence of viable cells.

18. The method of claim 16, wherein a decrease in the number of cells in
the cell viability assay and/or a decrease in the luciferase activity
determines that the one or more candidate nephroprotectant compounds are
not nephroprotectants.

20. A method for determining the nephrotoxicity of one or more test
compounds, said method comprising:(i) contacting discrete populations of
LLC-PK1 cells with one or more test compounds;wherein a plurality of
different concentrations for each of said one or more test compounds
contacts a separate discrete population of LLC-PK1 cells;(ii) determining
the level of an indicator of nephrotoxicity for each of said populations
of LLC-PK1 cells contacted in step (i) to produce a dose response curve
for each of the one or more test compounds, wherein said indicator
measures luciferase activity;(iii) determining the nephrotoxicity of each
of said one or more test compounds;(iv) contacting additional discrete
populations of LLC-PK1 cells with a plurality of concentrations of one or
more control nephrotoxic compounds;(v) determining the level of an
indicator of nephrotoxicity for each of said additionally contacted
populations of LLC-PK1 cells contacted in step (iv) to produce a dose
response curve for each of said one or more control nephrotoxic
compounds; and(vi) determining the nephrotoxicity of each of the one or
more control nephrotoxic compounds;wherein each of the populations of
LLC-PK1 cells are located in separate wells in a tissue culture device
comprising a plurality of wells.

Description:

CROSS-REFERENCE TO RELATED APPLICATION

[0001]This application claims the benefit under 35 U.S.C. § 119(e) of
U.S. Provisional Patent Application No. 61/032,745 filed Feb. 29, 2008,
where this provisional application is incorporated herein by reference in
its entirety.

BACKGROUND

[0002]1. Technical Field

[0003]The present invention is directed to methods of determining the
nephrotoxicity of compounds and methods of identifying nephroprotectants.

[0004]2. Description of the Art

[0005]Numerous drugs and other substances are known to be nephrotoxic and
can cause renal failure through a variety of mechanisms including direct
toxicity to the renal tubules, allergic interstitial nephritis, and
crystallization of the drug within the renal tubules. Nephrotoxic drugs
include anticancer agents such as cisplatin, methotrexate, and
doxyrubicin; non-steroidal anti-inflammatories (NSAIDS) (e.g., COX-2
inhibitors), antivirals (e.g., acyclovir, indinivir),
acetylcholinesterase inhibitors, angiotensin II receptor blockers (ARBs),
lithium, radiographic contrast media, and antibiotics (e.g.,
aminoglycosides, amphotericin).

[0006]Aminoglycoside antibiotics are the most commonly used antibiotics
worldwide in the treatment of Gram-negative bacterial infections.
However, aminoglycosides induce nephrotoxicity in 10-20% of therapeutic
courses. Several in vitro approaches have been used to evaluate
aminoglycoside toxicity. These assays have evolved as the mechanistic
understanding of aminoglycoside toxicity has developed. However, the
critical interactions underlying these mechanisms are complex and remain
under investigation (Mingeot-Leclerq et al., 1999). Without a clear
consensus on the molecular mechanism of aminoglycoside toxicity, devising
an assay that employs a single sub-cellular component is both important
and challenging.

[0007]Binding of a positively charged aminoglycoside to negatively charged
membrane phospholipids is thought to be part of the entry process into
both kidney proximal tubule cells and inner ear hair cells (Humes et al.,
1992, Sastrasinh et al., 1982). Schacht and coworkers used a model
phospholipid bilayer with a non-covalently associated fluorescent probe
to monitor binding of aminoglycosides (Au et al., 1986). This assay was
specific for phosphatidylinositol bisphosphate and was successfully used
to rank the several aminoglycoside toxicities in accordance with their
previously reported ototoxicity. Unfortunately, the technique requires
extensive purification of the samples and a dust-free environment, which
limits its utility as a screening platform.

[0008]Another likely player in the initial uptake of aminoglycosides into
target cells is megalin (Nagai et al., 2001). Megalin is a large receptor
glycoprotein for multiple physiologically important ligands, including
vitamins and hormones. Megalin expression is observed at the primary
sites of aminoglycoside toxicity (kidney and inner ear), as well as in
several other tissues. A key piece of evidence implicating megalin in
aminoglycoside uptake is that mice deficient in megalin function
accumulate significantly lower levels of gentamicin in the kidney
(Schmitz et al., 2002). The development of renal cell lines with stable
megalin expression was an initial example of cell-based aminoglycoside
nephrotoxicity assays that were more versatile than animal models and
potentially more relevant than in vitro cell-free systems (Nielsen et
al., 1998).

[0009]One example of a stable cell line expressing megalin is the porcine
kidney tubule cell line, LLC-PK1. The LLC-PK1 cell line has formed the
basis of several cell based nephrotoxicity assays. When grown to
confluence in cell culture, tight junctions are formed, and the cells
begin unidirectional transport of water and salts, which results in the
formation of "domes". Domes are sections of the monolayer that separate
from the culture dish and are visible using light microscopy. These domes
indicate healthy, functioning LLC-PK1 cells, and therefore, dome loss is
one measure of the extent of aminoglycoside toxicity. This toxicity
readout was validated by the discovery that a series of aminoglycosides
with different nephrotoxic potential in the rat model showed the same
rank order in their ability to degrade domes in LLC-PK1 culture (Takamoto
et al., 2002). Although the initial results with the LLC-PK1 dome loss
assay showed correlation with in vivo data of aminoglycoside activity,
the assay requires careful microscopic analysis of each sample, and thus,
this labor intensive method is not suitable for screening large numbers
of novel compounds.

[0010]Another LLC-PK1 assay was reported by Mingeot-Leclercq and coworkers
(Servais et al., 2005). Cultured LLC-PK1 cells were treated with
gentamicin, and the resulting apoptosis response was measured by various
spectrophotometric and staining methods. This more direct, quantitative
approach provided the opportunity to observe a dose-response in
gentamicin-induced toxicity, which may be an important capability when
attempting to distinguish closely related compounds. Subsequently, the
same group demonstrated that gentamicin caused a dose-dependent apoptosis
response at much lower gentamicin concentrations when the aminoglycoside
was introduced to the LLC-PK1 cells by electroporation (Servais et al.,
2006). However, this electroporation technique does not measure the
toxicity of aminoglycosides via their in vivo uptake route, which could
lead to an inaccurate estimation of their in vivo nephrotoxicity.

[0011]Drug-associated nephrotoxicity may be treated or prevented by the
administration of nephroprotectant compounds. Nephroprotectants are
compounds that ameliorate the toxicity induced by aminoglycosides and
other nephrotoxic compounds. An extensive body of literature describes
such candidate nephroprotectant compounds that afford protection against
aminoglycoside antibiotics. A non-limiting example of a list of
nephroprotectants includes aspirin, benzoic acid derivatives,
poly-phenols, and poly-anions. Much of the work testing candidate
nephroprotectants has been performed in animal models. However, using
animal models limits the number of compounds that can be studied and is
not suitable for screening large numbers of compounds.

[0012]One example of a well studied aminoglycoside nephroprotectant in
human clinical trials is aspirin. Patients treated concurrently with
intravenous gentamicin and oral aspirin suffered significantly less
hearing loss than a similar group of patients treated with gentamicin
alone (Sha et al., 2006). However, the aspirin treatment caused gastric
bleeding in some patients, which resulted in their removal from the
study. Even with this side effect, the toxicity prevention observed with
aspirin treatment may be useful, given that gentamicin-induced
ototoxicity can be debilitating and is generally irreversible. Aspirin is
thought to modulate aminoglycoside toxicity by acting as an anti-oxidant
and reversing the effects of reactive oxygen species (ROS) generated by
aminoglycoside exposure (Chen et al., 2006). However, aspirin does not
seemed extremely well suited for use as a nephroprotectant, because it
can cause both acute and chronic nephrotoxicity at high doses in humans
and experimental animals (Black 1986).

[0013]Additional examples in the art of aminoglycoside nephroprotectants
include other benzoic acid derivatives. One of these compounds,
2,3-dihydroxybenzoate (DHB), has shown a protective effect in both
aminoglycoside ototoxicity and nephrotoxicity animal models. In an
ototoxicity animal model, Song and colleagues found that DHB was an
effective co-therapy administered with gentamicin, which resulted in
significantly lower shifts in auditory thresholds in guinea pigs (Song et
al., 1996). In a rat nephrotoxicity model, treatment with DHB reduced
elevations in blood, urea, nitrogen (BUN) and serum creatinine after
gentamicin treatment for eight days (Walker et al., 1998). Unlike
aspirin, DHB appears to present a benign toxicity profile. As part of a
DHB safety and efficacy study for iron chelation treatment of
β-thalassemia, patients were treated with oral doses of DHB for one
year (Miller 1979). Although no toxicity was observed, DHB did not
exhibit the desired therapeutic efficacy and was not pursued further.

[0014]Studies performed by Graziano and colleagues revealed that DHB
inhibits H2O2 induced membrane peroxidation in erythrocytes,
adding further support to the proposal that ROS play a role in
aminoglycoside toxicity (Graziano et al., 1976). Additional studies
confirmed that DHB treatment provided only partial protection from
gentamicin nephrotoxicity in rats. In addition, DHB has poor solubility
in aqueous solution, which significantly limits its utility as a
co-therapy with aminoglycosides.

[0015]Polyphenolic natural products such as the putative anti-oxidants
resveratrol and curcumin have been investigated as nephroprotectants
(Silan et al., 2007; Farombi et al., 2006). In rat studies, these
compounds only attenuated gentamicin toxicity but did not prevent it. The
majority of compounds in this class have limited utility as
aminoglycoside nephroprotectants due to their poor aqueous solubility.

[0016]Lysozomal sequestration of aminoglycosides affords protection from
nephrotoxicity in rats (Williams et al., 1986). In particular,
poly-anions and polyaspartic acid have been shown to prevent
aminoglycoside binding to renal brush border membranes in vitro (Kishore
et al., 1990; Kishore et al., 1992). The interaction between positively
charged aminoglycosides and negatively charged polypeptides leads to
lysozomal accumulation of aminoglycosides. This mechanism may be more
effective than radical-scavenging, as it interferes with the
aminoglycoside toxicity response at an earlier stage. However, an
important caveat to using polyaspartic acid to direct the lysozomal
accumulation of aminoglycosides may be that nephrotoxicity is merely
delayed, rather than prevented. At the end of a 14 day treatment period
with gentamicin and/or polyaspartic acid, rats co-dosed with polyaspartic
acid showed a five-fold higher gentamicin concentration in their kidneys
compared to rats treated with gentamicin alone. However, after the two
week treatment period, subsequent monitoring of the rats revealed that
the all of the gentamicin was eliminated from the kidney over the
following 16 weeks, with no creatinine elevation or histopathological
changes in the co-dosed polyaspartic acid treated animals. These results
support the use of polyaspartic acid as a nephroprotectant against
aminoglycosides. Studies in both rats and dogs have suggested that
co-administration of polyaspartic acid may not interfere with
aminoglycoside antibiotic therapy (Reinhard et al., 1994; Whittem et al.,
1996).

[0017]In summary, while a variety of nephroprotectant compounds exist in
the art, many of these have proven to be of limited effectiveness in
preventing human nephrotoxicity and are associated with undesirable side
effects. Thus, there is a clear need for safe and effective
nephroprotectant compounds suitable for human use. Additionally, given
the significant dangers associated with the nephrotoxicity of numerous
drugs, there clearly remains a need to be able to determine the
nephrotoxicity of therapeutic compounds in order to safely treat human
infections and disease.

[0018]The present invention addresses these needs by providing novel
high-throughput methods for determining the nephrotoxicity of compounds
as well as for identifying novel nephroprotectant compounds. These in
vitro methods for determining compound nephrotoxicity correlate well with
in vivo nephrotoxicity, and are, therefore, suitable predictors of in
vivo nephrotoxicity.

BRIEF SUMMARY

[0019]The present invention is based on the development of a novel in
vitro assay of nephrotoxicity, which may be used to determine in vivo
nephrotoxicity of compounds and identify nephroprotectants.

[0020]In one embodiment, the present invention includes a method for
determining the nephrotoxicity of one or more test compounds, said method
comprising: (i) contacting discrete populations of human kidney
epithelial cells with one or more test compounds; wherein each of a
plurality of different concentrations for each of said one or more test
compounds contacts a separate discrete population of cells; (ii)
determining the level of an indicator of nephrotoxicity for each of said
populations of contacted cells of step (i) in order to produce a dose
response curve for each of the one or more test compounds; and (iii)
determining the nephrotoxicity of each of said one or more test
compounds. In one embodiment, the cells are human, and in a related
embodiment, the cells are HK-2 cells.

[0021]In one embodiment, the present invention includes a method for
determining the nephrotoxicity of one or more test compounds or a
combination of test compounds, said method comprising: (i) contacting
discrete populations of human kidney epithelial cells with one or more
test compounds or compound mixtures; wherein each of a plurality of
different concentrations for each of said one or more test compounds or
compound mixtures contacts a separate discrete population of cells; (ii)
determining the level of an indicator of nephrotoxicity for each of said
populations of contacted cells of step (i) in order to produce a dose
response curve for each of the one or more test compounds; and (iii)
determining the nephrotoxicity of each of said one or more test
compounds. In one embodiment, the cells are human, and in a related
embodiment, the cells are HK-2 cells.

[0022]In related embodiments, each of the populations of cells is located
in separate wells in a tissue culture device comprising a plurality of
wells. In particular embodiments, the plurality of wells is selected from
the group consisting of 4, 6, 12, 24, 48, 96, 384, and 1536 wells.

[0023]In another embodiment, the above method for determining the
nephrotoxicity of one or more test compounds, further comprises: (iv)
contacting additional discrete populations of kidney epithelial cells
with a plurality of concentrations of one or more control nephrotoxic
compounds; (v) determining the level of an indicator of nephrotoxicity
for each of said additionally contacted populations of cells of step
(iv), in order to produce a dose response curve for each of said one or
more control nephrotoxic compounds in said additionally contacted
populations of cells of step (iv); and (vi) determining the
nephrotoxicity of each of the one or more control nephrotoxic compounds.

[0024]In related embodiments, the plurality of concentrations of the one
or more test compounds tested and the plurality of concentrations of the
one or more control nephrotoxic compounds tested are both in the range of
about 1 mg/mL to about 1 ug/mL.

[0025]In another related embodiment, the determination of the
nephrotoxicity of each of said one or more compounds comprises
calculating an EC50 for each of the one or more test compounds from the
dose response curves of step (ii).

[0026]In further related embodiments, the determination of the
nephrotoxicity of each of said one or more compounds comprises comparing
the EC50 for each of the one or more test compounds to an EC50 for each
of the one or more nephrotoxic compounds calculated from the dose
response curves from step (iv).

[0027]In yet other related embodiments, the one or more control
nephrotoxic compounds are selected from the group consisting of:
amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,
streptomycin, tobramycin, apramycin.

[0028]In particular embodiments, the one or more test compounds are
aminoglycosides.

[0029]In some embodiments, the indicator of nephrotoxicity is apoptosis.

[0030]In further embodiments, the indicator of nephrotoxicity is caspase
activity.

[0031]In yet other embodiments, the caspase activity is caspase-3
activity.

[0032]In specific embodiments, the caspase-3 activity is determined using
a bioluminescent substrate.

[0033]A further related embodiment of the present invention is a method
for determining the in vivo nephrotoxicity of a test compound, said
method comprising: (i) determining the EC50 of the test compound and one
or more control nephrotoxic compounds using an in vitro kidney epithelial
cell assay; (ii) comparing the EC50 of the test compound to the EC50 of
one or more control nephrotoxic compounds, wherein the EC50 of the one or
more in vivo nephrotoxic compounds correlates with an in vivo indicator
of nephrotoxicity, thereby determining the in vivo nephrotoxicity of a
test compound.

[0034]In related embodiments, the in vivo indicator of nephrotoxicity is
selected from the group consisting of: an increase in BUN levels,
increased serum creatinine levels, and caspase activity.

[0035]In further related embodiments, the in vivo indicator of
nephrotoxicity is an increase in BUN levels.

[0036]In another embodiment, the present invention includes a method for
determining the ability of one or more candidate nephroprotectant
compounds to act as a nephroprotectant, said method comprising: (i)
contacting discrete populations of HK-2 cells with a plurality of
different concentrations of one or more candidate nephroprotectant
compounds in the presence of a nephrotoxic compound, wherein each of a
plurality of different concentrations for each of said one or more
candidate nephroprotectant compounds contacts a separate discrete
population of HK-2 cells; (ii) determining the levels of an indicator of
nephrotoxicity for each one of the contacted populations of HK-2 cells of
step (i); and (iii) determining the ability of each one or more candidate
nephroprotectant compounds to act as a nephroprotectant.

[0037]In a further related embodiment, the above method further comprises:
(iv) contacting additional discrete populations of HK-2 cells with a
plurality of different concentrations of said one or more candidate
nephroprotectant compounds in the absence of a nephrotoxic compound,
wherein each of a plurality of different concentrations for each of said
one or more candidate nephroprotectant compounds contacts a separate
discrete population of HK-2 cells, wherein the ability of a candidate
nephroprotectant compound to act as a nephroprotectant is determined when
a dose-dependent decrease in the indicator of nephrotoxicity is present
in the contacted HK-2 cells of step (i) compared to the indicator of
nephrotoxicity in the contacted HK-2 cells of step (iii) for a given
candidate nephroprotectant compound.

[0038]In other embodiments, the present invention includes a method for
determining the ability of one or more candidate nephroprotectant
compounds to act as a nephroprotectant, said method comprising: (i)
contacting discrete populations of kidney epithelial cells with a
plurality of different concentrations of said one or more candidate
nephroprotectant compounds, wherein each of a plurality of different
concentrations for each of said one or more test compounds contacts a
separate discrete population of cells; (ii) contacting additional
discrete populations of cells with a plurality of different
concentrations of said one or more candidate nephroprotectant compounds
as in step (i), and further contacting all the additional discrete
populations of cells with a static concentration of nephrotoxic compound;
(iii) determining the levels of an indicator of nephrotoxicity for each
one of the contacted populations of cells of step (i) and step (ii); and
(iv) determining the ability of each one or more candidate
nephroprotectant compounds to act as a nephroprotectant.

[0039]In related embodiments, a dose-dependent decrease present in the
indicator of nephrotoxicity in the contacted cells of step (iii) compared
to the indicator of nephrotoxicity in the contacted cells of step (ii)
for a given candidate nephroprotectant compound; and wherein the
contacted cells of step (ii) and step (iii) have the same concentration
of the given candidate nephroprotectant compound determines the given
candidate nephroprotectant compound's ability to act as a
nephroprotectant.

[0040]In some embodiments, the indicator of nephrotoxicity is apoptosis.
In particular embodiments, the indicator of nephrotoxicity is caspase
activity. In further embodiments, the caspase activity is caspase-3
activity.

[0041]In another embodiment, the method for determining the ability of one
or more candidate nephroprotectant compounds to act as a nephroprotectant
further comprises: (v) validating the ability of said one or more
candidate nephroprotectant compounds to act as a nephroprotectant by
performing one or more counterscreens.

[0042]In further embodiments, said one or more counterscreens are selected
from the group consisting of: a cell viability assay, and an assay for
luciferase activity. In particular embodiments, the counterscreens are a
cell viability assay followed by and an assay for luciferase activity. In
another particular embodiment, the luciferase activity is produced in the
presence of viable cells.

[0043]In certain embodiments a decrease in the number of cells in the cell
viability assay and/or a decrease in the luciferase activity determines
that the one or more candidate nephroprotectant compounds are not
nephroprotectants.

[0045]In yet another related embodiment, the present invention includes
kits for determining the nephrotoxicity of a candidate nephrotoxic
compound comprising: (i) instructions for using the kit; (ii) a
multi-well culture vessel of about 24, 48, 96, 384, or 1536 wells; (iii)
a luciferase substrate specific for caspase activity; and (iv) a control
nephrotoxic compound. In related embodiments the kits include kidney
epithelial cells, e.g., HK-2 cells. In other related embodiments, the
candidate nephrotoxic compound is selected from the group consisting of
aminoglycosides and aminoglycoside antibiotics.

[0046]In a further embodiment, the present invention also includes kits
for determining the ability of a candidate nephroprotectant compound to
act as a nephroprotectant comprising: (i) instructions for using the kit;
(ii) a multi-well culture vessel of about 24, 48, 96, 384, or 1536 wells;
(iii) a luciferase substrate specific for caspase activity; (iv) a
control nephrotoxic compound; (v) a cell viability determining reagent;
and (vi) luciferase conditional on cell viability. In related
embodiments, the kits further comprise kidney epithelial cells, e.g.,
HK-2 cells.

[0048]FIG. 1 is a schematic outline of the HK-2 in vitro nephrotoxicity
assay. The compound corresponding to the right-most line in the graph is
predicted to be more nephrotoxic than the compound corresponding to the
left-most line.

[0049]FIG. 2 is a graph showing results obtained for amikacin and
gentamicin in the HK-2 in vitro nephrotoxicity assay. Compounds were run
in duplicate with the high and the low values indicated as error bars.

[0050]FIG. 3 provides graphs comparing the correlation of different in
vitro nephrotoxicity assays with the in vivo nephrotoxicity of various
compounds. FIG. 3A is a graph showing a significant correlation between
the results of the HK-2 in vitro nephrotoxicity assay and the in vivo
14-day rat study conducted with amikacin, apramycin, gentamicin,
neomycin, compound A, and compound B. FIG. 3B is a graph showing poor
correlation between the results of the LLC-PK1 in vitro nephrotoxicity
assay and the in vivo 14-day rat study conducted with amikacin,
apramycin, gentamicin, neomycin, compound A, and compound B.

[0051]FIG. 4 is a graph showing a correlation between apoptosis readout of
an in vitro LLC-PK1 nephrotoxicity assay for a group of test compounds
and the relative nephrotoxicity of these compounds established in a
14-day rat study.

[0052]FIG. 5 is a graph showing the results of an in vivo HK-2
nephrotoxicity assay performed in the presence of various concentrations
of an aminoglycoside-dependent candidate nephroprotectant compound, in
the presence or absence of the aminoglycoside. The results show
concentration dependent reduction in the gentamicin-induced caspase
signal.

[0053]FIG. 6 is a flowchart of counterscreens used to evaluate potential
nephroprotectants.

DETAILED DESCRIPTION

[0054]The present invention is based, in part, upon the identification of
an in vitro assay for determining the nephrotoxicity of a compound. As
used herein, the term "nephrotoxic" describes a compound or an effect of
a compound that is damaging or toxic to the kidney. Nephrotoxic injury
can lead to acute renal failure, in which the kidneys suddenly lose their
ability to function, or chronic renal failure, in which kidney function
slowly deteriorates.

[0055]The results obtained using the assays described herein correlate
well with in vivo indicators of nephrotoxicity. Thus, the present in
vitro assay may be used to determine or predict the in vivo
nephrotoxicity of a compound. Moreover, the level of in vitro
nephrotoxicity for compounds assayed by the methods described herein
correlates well with the relative in vivo nephrotoxicity among those
compounds. In addition, the present assays are amenable to
high-throughput methods, and may be performed using automated devices to
evaluate multiple test compounds simultaneously.

[0056]In various embodiments, the methods of the present invention may,
therefore, be utilized to determine the in vitro or in vivo
nephrotoxicity of one or more compounds, to predict the in vivo
nephrotoxicity of one or more compounds, to predict the relative in vivo
nephrotoxicity among a group of compounds, and to screen one or more
compounds to identify nephrotoxic compounds.

[0057]One of skill in the art would appreciate that the nephrotoxicity for
mixtures of compounds can be different from or the same as the aggregate
individual compound nephrotoxicity. Thus, as the therapeutic benefits
achieved from mixtures of compounds (e.g., aminoglycosides) can outweigh
the combined nephrotoxicity of said mixtures, embodiments of the present
invention are suitable to determine the nephrotoxicity of a compound
mixture.

[0058]Many of the previous studies in the art have examined the
nephrotoxicity of compounds in a piecemeal fashion with a variety of
dissimilar assays. The present invention addresses the lack of standard
format, large scale methods for determining the nephrotoxicity of test
compounds. By rapidly screening a plurality of test compounds using the
in vitro methods of the present invention, the skilled artisan can
readily identify more suitable therapeutic compounds (i.e., with lower
indices of nephrotoxicity), which may be chosen to safely and effectively
treat human disease and infection.

[0059]This is particularly relevant for the identification of
aminoglycoside antibiotics. As used herein, the term "aminoglycoside"
means an antibiotic compound that is characterized by the presence of an
aminocyclitol ring linked to an aminosugar in its structure. This group
of antibiotics is effective against aerobic and facultative aerobic
Gram-negative bacilli and some Gram-positive bacteria such as
staphylococci. A non-limiting list of commonly known aminoglycosides
includes: amikacin, gentamicin, kanamycin, neomycin, netilmicin,
paromomycin, streptomycin, tobramycin, apramycin, and modified
derivatives thereof. Aminoglycoside antibiotics are the most common form
of treatment for gram negative bacterial infections in the world. Yet,
the use of aminoglycoside antibiotics to treat such infections results in
nephrotoxicity in 10-20% of the cases. Thus, high-throughput, in vitro
assays designed to identify aminoglycoside antibiotics with relatively
low inherent nephrotoxicity are beneficial in both treating bacterial
infections and reducing the health care costs covering the treatment of
kidney damage. Accordingly, in particular embodiments, compounds assayed
by the methods of the present invention are aminoglycoside antibiotics.

[0060]In addition, in various embodiments, the methods of the present
invention may be utilized to identify nephroprotectant compounds. As used
herein, the term "nephroprotectant" means a compound or agent that is
able to ameliorate, reduce, inhibit, or prevent the damaging or toxic
effects induced by a nephrotoxic compound. Since the present assays may
be performed in a high-throughput manner, the assays can be used to
simultaneously measure the ability of one or more compounds to act as
nephroprotectant compounds against a broad spectrum of nephrotoxic
compounds or against specific nephrotoxic compounds, such as
aminoglycosides. In various embodiments, the methods of present
invention, therefore, may be used to screen for the ability of one or
more compounds to act as a nephroprotectant; ameliorate, prevent,
inhibit, or reduce the nephrotoxicity of a compound; and/or function as
an inhibitor of caspase activity, thereby resulting in a nephroprotective
effect.

[0061]One of skill in the art would understand that mixtures or
combinations of compounds can provide a level of nephroprotection that is
different from or the same as the levels of aggregate individual compound
nephroprotection. Thus, in certain embodiments, methods of the present
invention are suitable and used to determine the level of
nephroprotection of a compound mixture.

[0062]In certain embodiments, methods of the present invention are
practiced using HK-2 cells or LL-PK1 cells. HK-2 cells are a human kidney
epithelial cell line, available from American Type Culture Collection
(ATCC; CRL-2190). As described in the accompanying Examples, assays
performed using these cells resulted in nephrotoxicity values that
closely correlated with in vivo indicators of nephrotoxicity, while
LLC-PK1 cells, a porcine kidney tubule cell line, produced nephrotoxicity
values that correlated well with the rank order of in vivo nephrotoxicity
among different compounds. Accordingly, in particular embodiments, cells
utilized according to the present invention are any kidney epithelial
cells or kidney tubule cells. These cells may be derived from primary
cells or may be from a cell line. The cells may be obtained from any
mammalian source that is amenable to primary culture and/or adaptation
into cell lines. In lieu of generating cell lines from animals, such cell
lines may be obtained from, for example, American Type Culture
Collection, (ATCC, Rockville, Md.), or any other Budapest Treaty or other
biological depository. In one embodiment, the cells are derived from
humans or other primates, rats, mice, rabbits, sheep, dogs, and the like.
In one preferred embodiment, the cells are human kidney epithelial cells.
In certain embodiments, the cells are porcine kidney tubule cells.

[0063]Techniques employed in mammalian primary cell culture and cell line
cultures are well known to those of ordinary skill in that art. Indeed,
commercially available cell lines are generally accompanied by specific
directions for culturing cells in preferred growth conditions, along with
particular media formulations that are optimized for that given cell
line. The cells may be cultured or grown in any suitable growth media.
The media may optionally be serum-free. In one particular embodiment, the
cells are cultured in media comprising one or more growth factors, such
as, epidermal growth factor (EGF) and/or bovine pituitary extract (BPE).
In one embodiment, growth media comprising EGF and/or BPE are not
filtered subsequent to the addition of EGF and/or BPE to the media.
Additional growth media and conditions are described in the accompanying
Examples.

[0064]Certain embodiments of the present in vitro methods are conducted in
the form of high-throughput assays. As used herein, the term
"high-throughput" means a process for assaying multiple samples for a
desired biological activity or property. High-throughput assays are
generally automated, and may allow for assaying multiple samples at the
same time. In particular embodiments, the methods are performed using a
plate with a plurality of wells or concavities, a plurality of which
contain discrete cell populations, therefore allowing individual assays
to be conducted. Plates used in high-throughput assays generally have 6,
12, 24, 48, 96, 384, or 1536 wells.

[0065]The use of a high-throughput platform allows for the standardization
of assay conditions within a given set of experiments. High-throughput
assays also reduce the experimental variation, both within and between
experiments, as much of the assay can be automated. These features also
lead to more meaningful comparisons of data among different trials. Other
advantages of using in vitro high-throughput assays are that they
generate far more data in a shorter time-frame. Moreover, performing the
assays becomes are less labor intensive, more cost effective, and yield
more reproducible results than in vitro assays conducted in a more
piecemeal fashion.

[0066]It would be understood by one of ordinary skill in the art that any
of the in vitro methods described herein are amenable to a
high-throughput format, wherein the nephrotoxicity of a plurality of
compounds or compound mixtures (and/or concentrations thereof) or the
ability of a plurality of compounds or compound mixtures to act as
nephroprotectants is assayed at a plurality of different concentrations.

A. Determining Nephrotoxicity of a Compound

[0067]In one embodiment, the present invention provides a method of
determining the nephrotoxicity of a test compound, comprising contacting
kidney epithelial cells, e.g., HK-2 cells, with the test compound, and
determining the level of an indicator of nephrotoxicity, wherein the
level of the indicator is indicative of nephrotoxicity. In particular
embodiments, the nephrotoxicity of the test compound is determined by
comparing the level of the indicator to a control value. The control
value may be a predetermined value based upon values obtained using one
or more known nephrotoxic compounds, or it may be a control value
determined using one or more known nephrotoxic compounds. Typically, at
the same time, the test compound is being evaluated for nephrotoxicity.

[0068]In another embodiment, methods of the present invention measure the
nephrotoxicity for a mixture of test compounds.

[0069]In a related embodiment, the methods of the present invention are
used to determine or predict the in vivo nephrotoxicity of a test
compound, by comparing the level of the indicator of nephrotoxicity
produced by the test compound to the level(s) of the indicator of
nephrotoxicity produced in the same in vitro assay by one or more known
nephrotoxic compounds having a known in vivo nephrotoxicity. By comparing
or correlating the level produced by the test compound to the level
produced by one or more known nephrotoxic compounds, the in vivo
nephrotoxicity of the test compound may be readily determined.

[0070]In particular embodiments, the levels of in vitro nephrotoxicity of
a test compound are used to rank the nephrotoxicity of said test compound
among one or more nephrotoxic compounds whose nephrotoxicity is known or
that has been previously established by methods of the present invention.

[0071]A variety of different molecules may be employed as indicators of
nephrotoxicity in the in vitro assays of the present invention. In
particular embodiments, an indicator of nephrotoxicity is a measure of
cell injury. In related embodiments damage to kidney cells is monitored
by the release of brush border enzymes. In other embodiments, changes in
kidney cell gene expression in genes such as osteopontin, inositol
polyphospate multikinase, I-arginine glycine amidinotransferase,
prosaposin, lipocalin, synaptogyrin 2, kallikrein, KIM-1, kidney injury
molecule 1 (Kim1), lipocalin 2 (Lcn2) can be used to indicate
nephrotoxicity of a compound. Examples of other genes that whose
expression levels can be measure to indicate nephrotoxicity of a compound
are describe in Amin et al., Environ Health Perspect. 2004 March;
112(4):465-79 and Wang et al., Toxicology. 2008 Jan. 16 [Epub ahead of
print; PMID: 18289764], herein incorporated by reference in their
entirety. One having ordinary skill in the art would appreciate that a
variety of methods can be used to measure changes in gene expression, for
example, DNA slot blot, RT-PCR, real-time PCR, oligonucleotide and cDNA
microarrays, primer extension, S1 nuclease assay, and RNAse protection
assays, among others.

[0072]In other embodiments of the present invention, the indicator of in
vitro nephrotoxicity is a measure of cell death. The nephrotoxic effects
of test compounds, and more particularly, aminoglycosides, may occur via
several possible mechanisms, including inhibition of protein synthesis,
mitochondrial injury, and DNA damage. These cellular insults ultimately
lead to activation of programmed cell death pathways and apoptosis. Thus,
indicators of in vitro nephrotoxicity suitable for use with the present
invention are indicators of apoptosis. Indicators of apoptosis can be
characterized by distinct morphologic changes consisting of cell
shrinkage, nuclear condensation, and internucleosomal DNA fragmentation.

[0073]Preferably, in one embodiment, the in vitro indicator of
nephrotoxicity is a biochemical indicator of apoptosis. For example,
biochemical indicators of apoptosis may monitor caspase 8 activity, which
is induced predominantly from apoptotic stimuli received via integral
membrane death receptors such as Fas and TNFR1.

[0074]In another embodiment, biochemical indicators of apoptosis initiated
from mitochondria may be assayed, such as caspase-9 activity or
cytochrome C release. Alternatively, rather than monitoring apoptosis
from a single pathway, a particular embodiment monitors the common
effector of different apoptosis pathways, for example, caspase-3
activity. It has been established in the art that once activated, both
caspases 8 and 9 participate in a cascade that culminates in the
activation of caspase 3, which cleaves several substrates, resulting in
chromosomal DNA fragmentation and cellular morphologic changes
characteristic of apoptosis. Thus, it would be understood by one
ordinarily skilled in the art that when the in vitro indicator of
nephrotoxicity monitors caspase-3 activity, effectively all apoptotic
pathway are being monitored.

[0075]In one embodiment, the indicator of in vitro nephrotoxicity is a
morphological or biochemical indicator of apoptosis. In a particular
embodiment, the indicator of in vitro nephrotoxicity is a biochemical
marker of apoptosis such as cytochrome C oxidase activity, and caspase
activity.

[0076]In a more particular embodiment, the caspase activity for use as an
in vitro indicator of nephrotoxicity is selected from the group
consisting of: caspase-9 activity, caspase-8 activity, caspase-7
activity, and caspase-3 activity. In another embodiment, the activities
of one or more caspases selected from group consisting of caspase-9
activity, caspase-8 activity, caspase-7 activity, and caspase-3 activity
are used as indicators of in vitro nephrotoxicity. In yet a more
particular embodiment, the in vitro indicator of nephrotoxicity is
caspase-3 activity.

[0077]In related embodiments, caspase activity is determined by using a
conditionally activated luciferase substrate, wherein caspase cleavage of
the luciferase substrate makes the substrate available to luciferase. In
a particular embodiment, the luciferase substrate is specific for
measuring caspase-3 activity. Such assays are provided by Promega under
the name CASPASEGLO, which is a commercially available assay kit for
monitoring caspase activity based upon luminescence.

[0078]The skilled artisan would appreciate that many other enzymes
activated in apoptosis, and which are known in the art are equally suited
for use as an in vitro indicator of nephrotoxicity in the in vitro
nephrotoxicity assays described herein.

[0079]In various embodiments, the level of an in vitro indicator of
nephrotoxicity is measured continuously. The levels of in vitro
indicators of nephrotoxicity can be monitored at the time the compound or
mixture of compounds are added to a culture of cells of the present
invention. Monitoring can be done periodically, for example, at about 10
minutes, about 30 minutes, about 1 hour, about 2 hours, about 5 hours,
about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36
hours, about 48 hours, about 56 hours, about 64 hours, about 72 hours or
more after the compound or mixture of compounds are added to a culture of
cells of the present invention. Monitoring can also be performed at a
time between 0 hours and about 96 hours, 0 hours and about 84 hours, 0
hours and about 72 hours, 0 hours and about 60 hours, 0 hours and about
48 hours, 0 hours and about 36 hours, 0 hours and about 30 hours, or 0
hours and about 24 hours.

[0080]In particular embodiments, methods of the present invention comprise
determining a dose response curve and/or an EC50 of a test compound or
compound mixture. As used herein, the term "dose response curve"
describes a relationship between the amount of a compound or mixture
assayed and the resulting measured response. The term "dose" is commonly
used to indicate the amount of the compound or mixture used in the
experiment, while the term "response" refers to the measurable effect of
the compound or compound mixture being tested. Dose-response
relationships are determined graphically by plotting the varying compound
or mixture concentration on the X-axis in log scale and the measurable
response on the Y-axis. As used herein, the term "EC50" means the
concentration of a compound or mixture of compounds that induces a
response halfway between the baseline response and the maximum response
of that compound or compound mixture.

[0081]In related embodiments, the nephrotoxicity of a test compound is
determined by comparing the EC50 determined for a test compound by an in
vitro assay of the present invention to the EC50 of one or more known
nephrotoxic compounds, as determined using an in vitro assay of the
present invention.

[0082]In other related embodiments, the nephrotoxicity of a test compound
mixture is determined by comparing the EC50 determined for a test
compound mixture by an in vitro assay of the present invention to the
EC50 of one or more known nephrotoxic compounds or compound mixtures, as
determined using an in vitro assay of the present invention. Generally, a
dose-response curve for the test compound is determined by measuring the
level of an indicator of nephrotoxicity produced using various
concentrations of a test compound, such as a set of serial dilutions of
the test compound. The goal of determining the nephrotoxicity of a test
compound over a serially diluted concentration range is to provide for
the construction of a dose response curve. The X-axis of a dose response
curve generally represents the concentration of the test compound on a
log scale, whereas the Y-axis represents the response of the in vitro
indicator of nephrotoxicity to a particular concentration of test
compound.

[0083]Those skilled in the art would understand that a standard
dose-response curve is generally defined by four parameters: the baseline
response, wherein the indicator of nephrotoxicity does not increase above
the lowest concentration of test compound tested; the maximum response,
wherein there is no additional increase in the in vitro indicator of
nephrotoxicity with increasing concentrations of test compound; the slope
of the curve, wherein changes in the in vitro indicator of nephrotoxicity
increase with increasing test compound concentrations; and the EC50,
wherein the concentration of test compound produces a half-maximal
response in the in vitro indicator of nephrotoxicity for that given
compound. More simply stated, the EC50 is the concentration of test
compound that provokes a response half-way between the baseline response
and maximum response.

[0084]Thus, the EC50 is a convenient measure of the inherent
nephrotoxicity of a test compound. In addition, it would be understood by
the skilled artisan that the relative nephrotoxicity of a test compound
to a control nephrotoxic compound may be determined by comparing the
EC50s of the test compound to the control nephrotoxic compound. Thus, a
test compound is said to have a relatively high level of nephrotoxicity
when the EC50 of the test compound is less than the EC50 of a control
nephrotoxic compound. Likewise, the test compound is said to have a
relatively low level of nephrotoxicity when the EC50 of the test compound
is greater than the EC50 of a control nephrotoxic compound. The skilled
artisan would understand that the methods of the present invention are
able to determine all degrees of relative nephrotoxicity of a test
compound to any given control nephrotoxic compound and that such methods
are not limited to the examples above.

[0085]In particular embodiments of the present in vitro assays, a
plurality of different concentrations of a test compound are assayed.
This is useful in establishing a dose response curve and determining the
EC50 for a test compound. In other embodiments, a plurality of different
test compounds is assayed at essentially the same time. This is
particularly useful in a screen to determine the nephrotoxicity of a
large number of test compounds, e.g., to identify those compounds that
are nephrotoxic and those compounds that are not nephrotoxic. In one
particular embodiment, a plurality of test compounds are each assayed at
a plurality of different concentration, e.g., in order to determine dose
response curves and/or EC50 values for multiple test compounds.

[0086]In certain embodiments, the methods described herein measure the
response of an in vitro indicator of nephrotoxicity to a plurality of
test compound and control nephrotoxic compound concentrations in order to
determine the nephrotoxicity of a test compound.

[0087]One aspect of the present invention provides methods that relate to
measuring the nephrotoxicity of a compound using high-throughput in vitro
assays. Nephrotoxicity determined by the in vitro methods described
herein correlate well with in vivo indicators of nephrotoxicity, and
thus, are reliable predictors of in vivo nephrotoxicity. In another
aspect of the present invention, methods that measure the in vitro
nephrotoxicity of a compound correlate well with the relative in vivo
nephrotoxicity among different compounds assayed in a 14-day rat model of
nephrotoxicity.

[0088]In one embodiment, a high-throughput method for determining the
nephrotoxicity of a test compound comprises the steps of culturing cells
in a plurality of wells of a tissue culture device; contacting the cells
of each well with a particular concentration of test compound, selected
from a plurality of test compound concentrations; determining an
indicator of nephrotoxicity for each one of the plurality of different
concentrations of the test compound in said contacted cells in order to
produce a dose response curve; and determining the nephrotoxicity of said
test compound, wherein the test compound may be any pharmaceutical
compound.

[0089]In one embodiment, a high-throughput method for determining the
nephrotoxicity of a test compound comprises the steps of culturing cells
in a plurality of wells of a tissue culture device; contacting the cells
of each well with a particular concentration of test compound or control
nephrotoxic compound, selected from a plurality of test compound and
control nephrotoxic compound concentrations; determining an indicator of
nephrotoxicity for each one of the plurality of different concentrations
of the test compound and the control nephrotoxic compounds in said
contacted cells in order to produce a dose response curve of both the
test compound and the control nephrotoxic compound; and determining the
nephrotoxicity of said test compound relative to said control nephrotoxic
compound.

[0090]In another embodiment, a high-throughput method for determining the
nephrotoxicity of one or more test compounds comprises the steps of
culturing cells in a plurality of wells of a tissue culture device;
contacting the cells of each well with a particular concentration of a
different test compound or control nephrotoxic compound(s), selected from
a plurality of test compound and control nephrotoxic compound
concentrations; determining an indicator of nephrotoxicity for each one
of the plurality of different concentrations of each one or more test
compound and control nephrotoxic compound(s) in said contacted cells in
order to produce a dose response curve of both the one or more test
compounds and the control nephrotoxic compound(s); and determining the
nephrotoxicity of said one or more test compounds relative to said
control nephrotoxic compound(s).

[0091]In another embodiment, the cells are cultured in a tissue culture
device having a plurality of wells, wherein the plurality of wells is
selected from the group consisting of 4, 6, 12, 24, 48, 96, 384, and 1536
wells. In another embodiment, the tissue culture device is a microtiter
plate having a plurality of wells. In a particular embodiment, the
microtiter plate may have 4, 6, 12, 24, 48, 96, 384, or 1536 wells. In a
more particular embodiment, the microtiter plate may have 24, 48, 96, or
384 wells. In a preferred embodiment, the microtiter plate has 48, 96, or
384 wells. In another preferred embodiment, the microtiter plate has a
plurality of wells selected from the group consisting of 24, 48, 96, and
384 wells.

[0092]In certain embodiments, cells of the present invention are seeded in
96 well plates in a volume of about 25 μl, 50 μl, 75 μl, 100
μl, 125 μl, 150 μl, 175 μl, or 200 μl. In particular
embodiments, the density of seeded cells can be from about 100 cells/mL
to 1,000,000 cells/mL, about 250 cells/mL to 100,000 cells/mL, about 500
cells/ml to 10,000 cells/mL, or about 1,000 cells/mL to 8,000 cells/mL.
In related embodiments, the density of seeded cells is about 100 cell/mL,
about 250 cell/mL, about 500 cell/mL, about 1000 cell/mL, about 2000
cell/mL, about 3000 cell/mL, about 4000 cell/mL, about 5000 cell/mL,
about 6000 cell/mL, about 7000 cell/mL, about 8000 cell/mL, about 9000
cell/mL, about 10,000 cell/mL, about 50,000 cell/mL, about 100,000
cell/mL, about 500,000 cell/mL, or about 1,000,000 cell/mL, or any
intervening density.

[0094]In one embodiment, the methods of the present invention determine
the nephrotoxicity of compound that is classified as an aminoglycoside.
In a particular embodiment, the compound assayed is an aminoglycoside
antibiotic selected from the group consisting of gentamicins, kanamycins,
streptomycins, amakicins, apramycins, netilmicins, paromomycins,
tobramycins, and modified derivatives thereof.

[0095]In a more particular embodiment, the inherent nephrotoxicity is
determined for a compound with either a known, suspected, or unknown
level of nephrotoxicity.

[0096]The skilled artisan would understand that the methods of the present
invention are particularly useful for establishing the nephrotoxicity of
known, suspected, or unknown nephrotoxic compounds, because the in vitro
methods described herein correlate well with in vivo indicators of
nephrotoxicity, whereas previous measures of the nephrotoxicity in the
art of such known, suspected, or unknown nephrotoxic compounds may not be
truly indicative of their in vivo nephrotoxicity. The skilled artisan
would also appreciate that in vitro assays of the present invention are
suitable for establishing the rank order of in vivo nephrotoxicity among
different known, suspected, or unknown nephrotoxic compounds.

[0097]In one embodiment, a high-throughput method for determining the
nephrotoxicity of a test compound comprises the steps of culturing cells
in a plurality of wells of a tissue culture device; contacting the cells
of each well with a particular concentration of test compound, selected
from a plurality of test compound concentrations; determining the levels
of an indicator of nephrotoxicity for each one of the plurality of
different concentrations of the test compound in said contacted cells in
order to produce a dose response curve; and determining the
nephrotoxicity of said test compound, wherein the plurality of
concentrations of the test compound is a serially diluted range of
concentrations within the range of about 0 μg/mL to about 1 mg/mL.

[0098]In one embodiment, in vitro nephrotoxicity is assayed for a
plurality of different concentrations of the test compound or compound
mixture with the goal of including some concentrations at which no toxic
effect is observed and also at least two or more higher concentrations at
which a toxic effect is observed. For example, assaying test compounds at
several concentrations within the range of about 0 μg/mL to about 1
mg/mL is commonly useful to achieve these goals. It will be possible or
even desirable to conduct certain of these assays at concentrations of
about 0.05 μg/mL to about 750 μg/mL, or of about 0.5 μg/mL to
about 500 μg/mL. In a particular embodiment, the range of
concentrations to be tested consists of a plurality of 2, 3, 4, 5, 6, 7,
8, 9, or 10 or more different concentrations within a range of about 0
μg/mL to about 1 mg/mL, or alternatively, within a concentration range
of about 0.5 μg/mL to about 500 μg/mL.

[0099]In a more particular embodiment, the plurality of different test
compounds or combinations of compounds are 2-fold serial dilutions,
3-fold serial dilutions, 4-fold serial dilutions, 5-fold serial
dilutions, 6-fold serial dilutions, 7-fold serial dilutions, 8-fold
serial dilutions, 9-fold serial dilutions, or 10-fold serial dilutions in
a range of concentrations of about 0 μg/mL to about 1 mg/mL, or
alternatively, within a concentration range of about 0.5 μg/mL to
about 500 μg/mL. For example, a series of ten, 2-fold serial dilutions
of a test compound starting at a concentration of about 500 μg/mL
would include test compound concentrations of about 500 μg/mL, about
250 μg/mL, about 125 μg/mL, about 62.5 μg/mL, about 31.3
μg/mL, about 15.6 μg/mL, about 7.8 μg/mL, about 3.9 μg/mL,
about 2 μg/mL, about 1 μg/mL, and about 0.5 μg/mL.

[0100]It would be understood by those skilled in the art that the
particular concentrations assayed in an in vitro nephrotoxicity assay of
the present invention may vary from one test compound to another.
Moreover, the concentrations assayed for a mixture of compounds varies
depending on the compounds in the mixture, and thus, mixtures of
compounds may contain lower, the same as, or greater concentrations of
compounds than would be present in an individual nephrotoxicity assay of
a compound in the mixture.

[0101]In one embodiment, the dose response curve of one or more test
compounds is compared to the dose response curves for one or more control
nephrotoxic compounds. In certain embodiments, the control nephrotoxic
compounds can be aminoglycosides, wherein the nephrotoxicity of said
control nephrotoxic compound is known. In a related embodiment the one or
more control nephrotoxic compounds can be aminoglycoside antibiotics. In
another related embodiment one or more control nephrotoxic compounds are
aminoglycoside antibiotics selected from the group of gentamicins,
kanamycins, streptomycins, amakicins, apramycins, netilmicins,
paromomycins, tobramycins, and modified derivatives thereof, wherein the
nephrotoxicity is known. In another embodiment, the one or more control
nephrotoxic compounds are aminoglycoside antibiotics selected from the
group of gentamicins, kanamycins, amakicins, and apramycins. In a
particular embodiment, the one or more control nephrotoxic compounds are
the aminoglycoside antibiotics gentamicin and amikacin.

[0102]It would be readily understood to those of skill in the art that
once a dose response curve is known for a particular test compound, that
test compound is capable of being utilized as a control nephrotoxic
compound. The skilled artisan would also recognize that the control
nephrotoxic compounds and test compounds should not be the same compound
in a given assay.

[0103]In a related embodiment, the relative nephrotoxicity of a test
compound or compound mixture is determined by comparing the dose response
curve for the test compound or mixture to the dose response curve for one
or more control nephrotoxic compounds.

[0104]An important feature of the novel methods of the present invention
described herein is the extremely high correlation between these in vitro
nephrotoxicity assay and the in vivo indicators of nephrotoxicity such as
blood, urea, nitrogen levels (BUN), serum creatinine levels, and
histopathological indicators of nephrotoxicity.

[0105]In particular embodiments, the in vitro indicator of nephrotoxicity
that measures apoptosis correlates well with one or more in vivo
indicators of nephrotoxicity, e.g., an in vivo indicator of
nephrotoxicity selected from the group consisting of BUN, serum
creatinine, and histopathology.

[0106]In more particular embodiments, the in vitro indicator of
nephrotoxicity measures caspase activity, which correlates well with the
minimum concentration of compound required in vivo to cause increases in
BUN and serum creatinine measured in a nephrotoxicity animal model. In
specific embodiments the indicator of in vitro nephrotoxicity is
caspase-3 activity, measured by luciferase, which correlates well with in
vivo increase in BUN levels in a 14 day nephrotoxicity rat model.

[0107]The skilled artisan would readily understand that "correlates well"
is a relative term and that many methods are known in the art to
calculate correlations. Furthermore, the skilled artisan is knowledgeable
regarding what constitutes a significant correlation and may make that
determination without further guidance provided herein. An in vitro EC50
of a test compound that correlates well with the minimum concentration of
test compound in vivo to cause an increase in an indicator of
nephrotoxicity can have a correlation of about 0.75 to about 1, about 0.8
to about 1, about 0.85 to about 1, about 0.9 to about 1, about 0.95 to
about 1 or any numerical value between 0.75 and 1.

[0108]For instance, as shown in FIG. 3, a nephrotoxicity assay performed
in HK-2 cells of control nephrotoxic compounds, gentamicin, amikacin,
apramycin, neomycin, and test compounds A and B correlates well with the
minimum concentrations of these compounds in vivo that cause increases in
BUN (FIG. 3A). In contrast, when the in vitro assay is carried out in
LLC-PK1 cells, gentamicin, amikacin, apramycin, neomycin, and test
compounds A and B fail to correlate with minimum concentrations of these
compounds in vivo that cause increases in BUN (FIG. 3B). However, as FIG.
4 clearly demonstrates, the in vitro nephrotoxicity measured for a group
of compounds in an LLC-PK1 assay correlates well with the relative in
vivo nephrotoxicity of these compounds in a 14-day rat nephrotoxicity
model.

B. Identification of Nephroprotectant Compounds

[0109]The methods described above may be readily adapted for the
identification of nephroprotectant compounds, including those compounds
that protect against specific nephrotoxic compounds or classes thereof,
and those that protect against multiple or all nephrotoxic compounds. The
art has failed to identify a sufficient number of useful nephroprotectant
compounds for human use. Moreover, there is currently insufficient data
regarding the ability of a single nephroprotectant to protect against a
plurality of nephrotoxic compounds.

[0110]In one embodiment, the present invention provides an assay to
determine the ability of a compound or combination of compounds to act as
a nephroprotectant, comprising contacting human kidney epithelial cells,
e.g., HK-2 cells, with a candidate nephroprotectant and a nephrotoxic
compound, and determining a level of nephrotoxicity as described above,
wherein a decrease in the level of nephrotoxicity indicates the candidate
nephroprotectant acts as a nephroprotectant against the nephrotoxic
compound. In another embodiment, one or more nephroprotectants are
assayed in parallel against a particular nephrotoxic compound or a group
of nephrotoxic compounds.

[0111]In particular embodiments, the present invention provides an assay
to determine the ability of a compound to act as a nephroprotectant,
comprising contact porcine kidney tubule cells, e.g., LLC-PK1 cells, with
a candidate nephroprotectant and a nephrotoxic compound, and determining
a level of nephrotoxicity as described above, wherein a decrease in the
level of nephrotoxicity indicates the candidate nephroprotectant acts as
a nephroprotectant against the nephrotoxic compound.

[0112]Certain embodiments of the present invention provide for large scale
high-throughput screening of small molecule libraries to identify
compounds with the ability to provide a nephroprotective effect. Some
advantages in using the high-throughput screens of the present invention
is that more nephroprotectant compounds can be identified, and the
nephroprotective effects of these compounds can be directly compared in a
single assay. Additionally, in certain embodiments, broad spectrum
nephroprotective compounds or mixtures can be identified by assaying the
nephroprotective effects of a compound against a variety of nephrotoxic
compounds.

[0113]In certain embodiments, cultured cells described elsewhere herein
are suitable for use with the present invention of determining the
ability of a compound to act as a nephroprotectant. For example, cells
may be primary kidney epithelial cell cultures or cultured kidney
epithelial cell lines. In particular embodiments, primate epithelial
kidney cells are well suited for the screening of nephroprotectant
compounds. In a specific embodiment, a human epithelial kidney cell line
such as HK-2 cells is the preferred cells to use in nephroprotectant
screens. In other embodiments, LLC-PK1 cells are used in nephroprotectant
screens.

[0114]In order to be characterized as a nephroprotectant compound,
increasing concentrations of the candidate nephroprotectant compound or
combination of compounds should elicit a dose dependent decrease in the
levels of the in vitro indicator of nephrotoxicity in the presence and/or
absence of the nephrotoxic compound.

[0115]In one embodiment screening for the ability of one or more candidate
nephroprotectant compounds or mixtures to act as a nephroprotectant,
comprises the steps of: (i) contacting discrete populations of HK-2 cells
with a plurality of different concentrations of said one or more
candidate nephroprotectant compounds, wherein each of a plurality of
different concentrations for each of said one or more test compounds
contacts a separate discrete population of HK-2 cells; (ii) contacting
additional discrete populations of HK-2 cells with a plurality of
different concentrations of said one or more candidate nephroprotectant
compounds as in step (i), and further contacting all the additional
discrete populations of HK-2 cells with a static concentration of
nephrotoxic compound; (iii) determining the levels of an indicator of
nephrotoxicity for each one of the contacted populations of HK-2 cells of
step (i) and step (ii); and (iv) determining the ability of each one or
more candidate nephroprotectant compounds to act as a nephroprotectant.

[0116]In further embodiments, an in vitro indicator of nephrotoxicity for
a nephrotoxic compound or compound mixture exhibits a dose dependent
signal reduction when exposed to increasing concentrations of candidate
nephroprotectant compound. In a related embodiment, the level of
apoptosis activity elicited by a static concentration of nephrotoxic
compound such as an aminoglycoside exhibits a dose dependent signal
decrease in response to an increasing concentration of candidate
nephroprotectant compound. In another related embodiment, the level of
caspase activity for a aminoglycoside antibiotic selected from the group
consisting of gentamicins, kanamycins, streptomycins, amakicins,
apramycins, netilmicins, paromomycins, tobramycins, and modified
derivatives thereof, exhibits a dose dependent signal decrease in
response to an increasing concentration of candidate nephroprotectant
compound.

[0117]In a specific embodiment, the level of caspase-3 activity for a
control aminoglycoside antibiotic such as gentamicin exhibits a dose
dependent signal decrease in response to an increasing concentration of
candidate nephroprotectant compound or mixture.

[0118]The skilled artisan would appreciate that when the candidate
nephroprotectant compound specifically reduces the apoptosis signal
caused by a nephrotoxic compound in a dose-dependent fashion, but without
altering the background apoptosis signal present in the absence of a
nephrotoxic compound, the nephroprotectant is specific nephrotoxic
compounds. In addition, if a candidate nephroprotectant compound causes a
dose dependent decrease in apoptosis signal elicited by one specific
nephrotoxic compound, (e.g., gentamicin), then the nephroprotectant is
specific for that nephrotoxic compound.

[0119]Alternatively, in a related embodiment, the candidate
nephroprotectant compound may reduce both the background levels of
apoptosis in the cultured cells lacking a nephrotoxic compound and the
apoptosis induced by the control nephrotoxic agent. This type of result
suggests that the candidate nephroprotectant compound acts on the
particular control nephrotoxic compound, as well as at a different point
in the apoptosis pathway.

[0120]In a particular embodiment, when the control nephrotoxic compound is
an aminoglycoside antibiotic, this dual mode of action by a given
candidate nephroprotectant compound should be relevant to enhancing the
clinical utility of aminoglycosides, since both the specific
aminoglycoside and the apoptosis response are blocked.

[0121]The skilled artisan would understand that many compounds may possess
biochemical activities sufficient to render them useful as
nephroprotectants, and thus, the present invention provides for screening
a wide range of candidate nephroprotectant compounds. As used herein, a
candidate nephroprotectant compound can be any chemical compound, for
example, a macromolecule (e.g., a polypeptide, a protein complex,
glycoprotein, or a nucleic acid) or a small molecule (e.g., an amino
acid, a nucleotide, an organic or inorganic compound).

[0122]A candidate nephroprotectant compound can have a formula weight of
less than about 10,000 grams per mole, less than 5,000 grams per mole,
less than 1,000 grams per mole, or less than about 500 grams per mole.

[0124]In particular embodiments, combinations or mixtures of the above
mentioned classes of nephroprotectant compounds are screened.

[0125]In certain embodiments the nephroprotectant compound screening assay
may additionally comprise the use of a control nephroprotectant compound.
It would be understood by one of skill in the art that once the
nephroprotective ability of a compound is known, that compound may be
suitable for use as a control nephroprotectant compound in the in vitro
assays of the present invention.

[0126]In another embodiment, the ability of a compound to act as a broad
spectrum nephroprotectant compound is assayed. In order to identify a
broad spectrum nephroprotectant compound, the ability of the
nephroprotectant compound would be assayed in the presence and absence of
1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or
more, 8 or more, 9 or more, or 10 or more nephrotoxic compounds.

[0127]For example, a particular candidate nephrotoxic compound can be
screened against various aminoglycoside antibiotics. In a particular
embodiment, the candidate nephroprotectant is screened for
nephroprotective ability against the aminoglycoside antibiotics selected
from the group consisting of gentamicins, kanamycins, streptomycins,
amakicins, apramycins, netilmicins, paromomycins, tobramycins, and
modified derivatives thereof, wherein the nephrotoxicity is known. In
another embodiment, the nephrotoxic compounds are aminoglycoside
antibiotics selected from the group of gentamicins, kanamycins,
amakicins, and apramycins. In a particular embodiment, the nephrotoxic
compounds are the aminoglycoside antibiotics gentamicin and amikacin.

[0128]The skilled artisan would appreciate that the more than one
candidate or combination of candidates can be screened at the same time
against a variety of nephrotoxic compounds as described above herein.

[0129]In one embodiment, libraries of small molecules can be screened for
the ability to act as a nephroprotectant against one or more nephrotoxic
compounds. A high-throughput screen for candidate nephroprotectant
compounds comprises the steps of culturing cells in a plurality of wells
of a tissue culture device; contacting the cells of each well with a
particular concentration of candidate nephroprotectant compound with or
without a static concentration of nephrotoxic compound, wherein the
candidate nephroprotectant compound concentration is selected from a
plurality different concentrations; detecting the levels of an indicator
of nephrotoxicity for each one of the plurality of different
concentrations of the candidate nephroprotectant compound in said
contacted cells either in the presence or absence of the static
concentration of nephrotoxic compound in order to identify the ability of
the candidate nephroprotectant compound to act as a nephroprotectant
against said nephrotoxic compound.

[0130]In related embodiments, a high-throughput screen for candidate
nephroprotectant compounds comprises the steps of culturing cells in a
plurality of wells of a tissue culture device; contacting the cells of
each well with a particular concentration of candidate nephroprotectant
compound with or without a static concentration of nephrotoxic compound,
wherein the candidate nephroprotectant compound concentration is selected
from a plurality different concentrations; monitoring an indicator of
nephrotoxicity for each one of the plurality of different concentrations
of the candidate nephroprotectant compound in said contacted cells either
in the presence or absence of the static concentration of nephrotoxic
compound in order to identify the ability of the candidate
nephroprotectant compound to act as a nephroprotectant against said
nephrotoxic compound, wherein the highest concentration of candidate
nephroprotectant compound tested is greater than the static concentration
of nephrotoxic compound.

[0131]In related embodiments, the candidate nephroprotectant compound is
assayed at a plurality of concentrations.

[0132]The highest concentration of candidate nephroprotectant compound
tested is about at least 50, about at least 40, about at least 30, about
at least 20 or about at least 10 times the concentration of control
nephrotoxic compound used in the assay.

[0133]The control nephrotoxic can be any compound wherein the dose
response curve for nephrotoxicity is known. The concentration of control
nephrotoxic compound used in the candidate nephroprotectant compound
assay is the concentration where there is about 90%, about 95%, 96%, 97%,
98%, 99%, or 100% nephrotoxicity, or alternatively, the concentration
necessary to initiate a uniform apoptosis response.

[0134]In one embodiment, the number of candidate nephroprotectant compound
concentrations assayed is about 20, about 15, about 10, about 5, or about
1, or any integer value between about 1 and about 20.

[0135]In a more particular embodiment, gentamicin is the control
nephrotoxic compound and is added to all assays at a concentration of 100
ug/mL.

[0136]In a related embodiment, the candidate nephroprotectant compound is
added at a concentration range of about 5000 ug/mL to about 0 ug/mL,
about 2500 ug/mL to about 1 ug/mL, or about 2000 ug/mL to about 2 ug/mL.

[0138]In various related embodiments, the level of an in vitro indicator
of nephrotoxicity measured in a nephroprotectant assay of the present
invention is measured continuously. In related embodiments, the ability
of a compound to act as a nephroprotectant can be monitored at the time
the nephroprotectant compound or mixture of compounds are added to a
culture of cells containing an aminoglycoside antibiotic. In particular
embodiments, monitoring can be done periodically, for example, at about
10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 5 hours,
about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36
hours, about 48 hours, about 56 hours, about 64 hours, about 72 hours or
more after the nephroprotectant compound or mixture of compounds are
added to a culture of cells containing an aminoglycoside antibiotic. In
related embodiments, monitoring can also be performed at a time between 0
hours and about 96 hours, 0 hours and about 84 hours, 0 hours and about
72 hours, 0 hours and about 60 hours, 0 hours and about 48 hours, 0 hours
and about 36 hours, 0 hours and about 30 hours, or 0 hours and about 24
hours.

[0139]The methods of the present invention also provide for
nephroprotectant validation assays or counterscreens. As used herein, the
term "counterscreen" means an assay that is used subsequent to an initial
screening assay to identify nephroprotectants. Counterscreens are
employed in order to validate and further describe the properties of
candidate molecules identified in screens as true positives. In some
instances, counterscreens are used to eliminate molecules identified in a
screening assay that possess undesired activities. Candidate
nephroprotectant compounds from an initial screen are subjected to a
series of counterscreens to discard any that interfere with the assay
readout rather than inhibit nephrotoxicity induced by a nephrotoxic
compound, such as an aminoglycoside (e.g., gentamicin).

[0140]One explanation for the source of a false positive result in an
initial nephroprotectant screen is a reduction in apoptosis (e.g.,
caspase activity) signal caused by the fact that the candidate
nephroprotectant itself is toxic to the cultured cells.

[0141]In one embodiment, a cell viability assay is used to rule out this
class of false positive, wherein the same concentrations of candidate
nephroprotectant compound are used in the presence of a viability marker.
Various viability markers may be used to determine cell viability. For
example, vital dyes such as azafloxin, basic blue (nile blue sulphate),
bismarck brown, basic red (rhodamine 6G), bengal red, brilliant crysyl
blue, eosin, fluorescein, gentian violet, indocyanine green, janus green,
methylene green, methylene blue, neutral red, trypan blue, trypan red,
and tetrazolium salts may be used in the methods of the present invention
to determine cell viability.

[0142]Alternatively, two other sources of false positives may be linked to
a particular embodiment of the present invention, wherein the in vitro
indicator of nephrotoxicity is a luciferase substrate that is
conditionally activated by caspase activity (i.e., a caspase/luciferase
substrate such as the CASPASEGLO reporter molecule). For example, in the
in vitro assays described herein, luminescence from a CASPASEGLO
substrate is only detected in the presence of both luciferase and caspase
enzymatic activities, and thus, a candidate nephroprotectant compound
that targets either luciferase or caspases would yield a false positive
result.

[0143]Although compounds that inhibit luciferase are rare, a second
counterscreen can be employed to eliminate this type of false positive.
In one embodiment luciferase activity is conditionally active in viable
cells, and thus, if the candidate nephroprotectant compound causes a
dose-dependent decrease in this assay, the candidate nephroprotectant
compound is likely to be specific for luciferase and can reliably be
detected as a false positive. For example, the fact that luciferase
requires ATP to produce bioluminescence from a luciferin substrate makes
for a luminescence measure that is conditionally active in viable cells,
as only viable cells generate ATP. Commercial kits to accomplish this
type of assay are available from Promega (e.g., CELLTITERGLO). In a
particular embodiment, the inhibition ATP dependent bioluminescence
elicited by a candidate nephroprotectant compound would suggest the
candidate nephroprotectant compound is not specific for a given
nephrotoxic compound but rather directly inhibits luciferase activity.

[0144]Another source of false positives, as mentioned above, would be
candidate nephroprotectant compounds that directly inhibit caspase
activity. In one embodiment, an in vitro counterscreen is employed that
determines the ability of the candidate nephroprotectant to inhibit the
cleavage of a caspase substrate. Perhaps the most convenient way to
measure caspase activity is by using conditionally active fluorogenic
caspase substrates. These molecules may comprise a fluorogenic molecule
such as aminomethylcoumarin (AMC), aminotrifluoromethylcoumarin (AFC),
rhodamine 110, GFP, and the like, and a caspase specific peptide, that
when cleaved will allow the fluorophore to become active. Many examples
of caspase specific peptides are known in the art. For example: YVAD is a
peptide specific for caspases 1 and 4; VDVAD is a peptide specific for
caspase 2; and DEVD is a peptide specific for caspases 3, 6, 7, 8, and
10. However, candidate nephroprotective molecules that inhibit caspases
need not be discarded, as caspase inhibitors would still act as
nephroprotectants to block nephrotoxicity, which is known in the art to
be mediated by apoptosis.

[0145]The in vitro assays of the present invention also provide reagents
that can comprise part of a kit. Such kits are useful in standardizing
assays to increase the reliability of results. Kits also present an
opportunity to decrease costs and increase the ease of manipulation
associated with the assays of the present invention.

[0146]In one embodiment, a kit is supplied for determining the
nephrotoxicity of a compound. Such kits comprise instructions for using
the kit, which are optionally accompanied by a table of previously
established dose response curves and EC50s of known nephrotoxic compounds
to facilitate the determination of nephrotoxicity of a compound. These
kits further comprise a multi-well culture vessel of about 24, 48, 96,
384 or 1536 wells. Such culture vessels can be microtiter plates, tissue
culture plates, slides, and the like. Kits also include an in vitro
indicator of nephrotoxicity, such as a luciferase substrate specific for
caspase activity and those described elsewhere herein. Kits may further
comprise one or more control nephrotoxic compounds. Kits optionally
comprise an epithelial kidney cell line, optionally the human epithelial
kidney cell line HK-2.

[0147]In another embodiment, a kit is supplied for determining the ability
of a candidate nephroprotectant compound to act as a nephroprotectant.
Such kits comprise instructions for using the kit; a multi-well culture
vessel of about 24, 48, 96, 384 or 1536 wells; a luciferase substrate
specific for caspase activity; a control nephrotoxic compound; and
reagents to be used in counterscreens. Counterscreening agents comprised
in the kit are cell viability determining reagents (e.g., vital dyes) and
luciferase assays conditional on cell viability, such as those described
herein. Optionally, kits may comprise a variety of well known
aminoglycosides, such as those discussed herein, to use in screening for
effective nephroprotectants. Kits optionally comprise an epithelial
kidney cell line, optionally the human epithelial kidney cell line HK-2.

[0148]In another embodiment, kits of the present invention comprise the
LLC-PK1 cell line or porcine kidney tubule cells.

[0149]While the terms used in the application are intended to be
interpreted with the ordinary meaning as understood by persons skilled in
the art, some terms are expressly defined in order to avoid ambiguity.

[0150]Prior to explaining at least one embodiment of the invention in
detail by way of exemplary figures, experimentation, results, and
laboratory procedures, it is to be understood that the invention is not
limited in its application to the details of construction and the
arrangement of the components set forth in the following description or
illustrated in the figures, experimentation and/or results. The invention
is capable of other embodiments or of being practiced or carried out in
various ways. As such, the language used herein is intended to be given
the broadest possible scope and meaning; and the embodiments are meant to
be exemplary--not exhaustive. Also, it is to be understood that the
phraseology and terminology employed herein is for the purpose of
description and should not be regarded as limiting.

EXAMPLES

Example 1

HK-2 Nephrotoxicity Assay

[0151]An in vitro nephrotoxicity assay was conducted using the human
kidney epithelial cell line, HK-2. An overview of the HK-2 nephrotoxicity
assay protocol is shown in FIG. 1.

[0152]HK-2 cells were cultured in KSFM, with 5 ng/mL epidermal growth
factor (EGF) and 0.05 mg/mL bovine pituitary extract (BPE). Cells were
maintained at sub-confluence and used to initiate the assay when they
reached 80% confluence. HK-2 cells were harvested using trypsin-EDTA and
dispensed into the wells of a 96-well polystyrene tissue-culture plate at
a density of 1.6×104 cells/well in a final volume of 100
uL/well. Plates were incubated at 37° C. with 5% CO2 for 3
days.

[0153]Compounds to be tested were diluted in KSFM with 10 mM HEPES buffer
that had been pre-warmed to 37° C. Plates were removed from the
incubator and the media was removed using a multichannel aspirator.
Diluted compounds were added to plates. The plates were subsequently
returned to the incubator overnight.

[0154]Plates were removed from the incubator and allowed to cool to room
temperature. 50 uL of CASPASEGLO (Promega) was added to each well. The
plates were briefly agitated on a shaker platform and then allowed to sit
at room temperature for 30 minutes. Luminescence was detected using a
plate reading luminometer. Increases in luminescence over the background
signal from untreated cells was indicative of apoptosis induced by the
addition of compound.

[0155]Novel compounds were usually tested in parallel with amikacin and
gentamicin controls on each plate. Typical control data is shown in FIG.
2. Gentamicin produced a full dose response curve over the concentration
range tested and an EC50 was calculated. As the concentration of
aminoglycoside increased, the luminescence signal corresponding to
induction of apoptosis also increased. Amikacin did not produce a maximal
response at the highest concentration tested (500 μg/mL), and
therefore, the EC50 could only be estimated.

[0156]In a pilot study, it was found that a majority of the novel
compounds screened had dose response curves distributed between
gentamicin and amikacin, such that EC50s could be calculated. The
apoptosis signal for some of the compounds screened was non-zero in the
absence of aminoglycoside. This background signal fluctuated with the
number of cells and was likely indicative of background levels of
apoptosis taking place in the cell culture.

[0157]In another study, more than 400 novel aminoglycosides were screened
in a high-throughput format for in vitro nephrotoxicity in HK-2 cells
using the methods described herein. Utilization of the high-throughput
format required orders of magnitude less compound (240 μg) than the
corresponding standard 14-day rat nephrotoxicity assay (6 g).

Example 2

Correlation of In Vitro HK-2 Assay with In Vivo Nephrotoxicity

[0158]To validate the utility of the HK-2 assay described in Example 1,
the nephrotoxicity of four commercial aminoglycoside antibiotics
(amikacin, gentamicin, neomycin and apramycin) and two other test
compounds (compounds A and B) were tested using both the in vitro HK-2
assay and a 14-day rat nephrotoxicity model. The rat in vivo assays
provided information in the form of acute toxicity, changes in Blood Urea
Nitrogen (BUN) and serum creatinine levels over time, as well as
histopathology changes in the kidneys of animals sacrificed at the end of
the study. Elevation in BUN or serum creatinine is one of the markers
routinely monitored in the clinic as an indication of aminoglycoside
nephrotoxicity.

[0159]These studies demonstrated a significant correlation between the
results from the in vitro HK-2 assay and the in vivo results obtained
from the 14-day rat nephrotoxicity model. Specifically, it was found that
the minimum concentration of compound that produced an increase in BUN in
the in vivo rat model correlated well with the EC50 from the in vitro
HK-2 assay (see FIG. 3A). Both assays indicated that amikacin was the
least nephrotoxic aminoglycoside tested, followed by apramycin,
gentamicin and neomycin, compound A, and compound B. The results obtained
with these six compounds suggested that both the in vitro HK-2 assay and
the in vivo rat model assay ranked the toxicity of the compounds in the
same order, and therefore, the in vitro HK-2 assay served as a suitable
predictor of in vivo nephrotoxicity established in the 14-day rat study.

[0160]In direct contrast, the correlation between the in vitro LLC-PK1
assay and the in vivo rat model was poor (FIG. 3B), and demonstrated that
the LLC-PK1 assay was not suitable to predict in vivo nephrotoxicity.
These results demonstrate that HK-2 cells represent a particularly useful
nephrotoxicity assay. Moreover, the results obtained using these
particular cells correlates well with in vivo nephrotoxicity.

Example 3

Correlation of In Vitro LLC-PK1 Assay with In Vivo Relative Nephrotoxicity

[0161]An in vitro nephrotoxicity assay was conducted using the porcine
kidney tubule cell line, LLC-PK1. Assays are conducted essentially as
described in Example 6, but with the modifications.

[0162]On Day 1 of the experiment, LLC-PK1 cells that had been maintained
in low glucose DMEM with 5% FBS were trypsinized and re-seeded at a
density of 8×103 cell/well in the wells of a 96-well
polystyrene tissue-culture plate. Plates were incubated at 37° C.
with 5% CO2 for 3 days.

[0163]On day 4, compounds to be tested were diluted in Ultraculture medium
(Cambrex) containing 2 mM L-glutamine and 20 mM HEPES buffer that had
been pre-warmed to 37° C. The compounds that were tested were
serially diluted from 400 μg/mL to 1 μg/mL. Plates were removed
from the incubator and the media was removed using a multichannel
aspirator. Cells were washed with Ultraculture medium and then the
diluted compounds were added to plates. The plates were subsequently
returned to the incubator overnight.

[0164]On day 5, plates were removed from the incubator and allowed to cool
to room temperature. 50 uL of CASPASEGLO (Promega) was added to each
well. The plates were briefly agitated on a shaker platform and then
allowed to sit at room temperature for 30 minutes. Luminescence was
detected using a plate reading luminometer. Increases in luminescence
over the background signal from untreated cells was indicative of
apoptosis induced by the addition of compound. Novel compounds were
usually tested in parallel with amikacin and gentamicin controls on each
plate. FIG. 4 shows the data generated from an in vitro LLC-PK1
experiment performed with Gentamicin, Kanamycin, Amikacin, and
Streptomycin correlated well with the relative nephrotoxicity of these
aminoglycosides in an in vivo 14-day rat nephrotoxicity study.

[0165]This study demonstrated a significant correlation between the
results from the in vitro LLC-PK1 assay and the relative in vivo
nephrotoxicity obtained from a 14-day rat nephrotoxicity model.
Specifically, it was found that the minimum concentration of compound
that produced an increase apoptosis in the in vivo rat model correlated
well with the levels of apoptosis measured in the in vitro LLC-PK1 assay
(see FIG. 4). Both assays indicated that streptomycin was the least
nephrotoxic aminoglycoside tested, followed by amikacin, neomycin, and
gentamicin. The results obtained with these four compounds suggested that
both the in vitro LLC-PK1 assay and the in vivo 14-day rat nephrotoxicity
model assay ranked the toxicity of the compounds in the same order, and
therefore, the in vitro LLC-PK1 assay served as a suitable predictor of
the relative in vivo nephrotoxicity of these compounds.

Example 4

Identification of Nephroprotectants

[0166]The nephrotoxicity assay was adapted to perform screens to identify
nephroprotectants. HK-2 cells were cultured as in Example 1. In a typical
example of a nephroprotectant screen, gentamicin was added to all wells
at 100 ug/mL to initiate a uniform apoptosis response. At the same time,
a dilution series of candidate nephroprotectants was added. The highest
nephroprotectant concentration tested was 20 times the gentamicin
concentration present in the assay. A dose-dependent reduction in the
gentamicin-induced apoptosis signal was suggestive of an additive that is
serving to block nephrotoxicity in those cells.

[0167]FIG. 5 shows an example of a potential nephroprotectant screened in
the in vitro assay. During screening both aminoglycoside-specific and
non-specific nephroprotectants was observed. The aminoglycoside-specific
compounds reduced the gentamicin induced apoptosis signal without
altering the background apoptosis signal. The second class of putative
nephroprotectants caused a reduction in both the gentamicin induced and
background apoptosis signals. This demonstrated that the two groups of
molecules acted at different points in the apoptosis response, both of
which are relevant to enhancing the clinical utility of aminoglycosides.

Example 5

Counterscreens of Candidate Nephroprotectants

[0168]This example describes various counterscreens that may be used to
validate candidate nephroprotectant compounds. Candidate
nephroprotectants from an initial screen were subjected to one of more
counterscreens to discard any that are interfering with the assay readout
rather than inhibiting gentamicin-induced toxicity. A schematic of this
process is shown in FIG. 6.

[0169]A trivial explanation for the reduction in caspase signal is that
the additives themselves are toxic to the cells. This was ruled out by a
standard viability assay (CELLTITERBLUE, Promega) run at the same
concentrations as the initial screen. The CASPASEGLO reporter used in the
in vitro assay provided a masked luciferase substrate that was activated
by caspase enzyme present in apoptotic cells. The activated substrate was
then available to a luciferase enzyme which was also included in the
reporter kit. The combination of caspase and luciferase activities
generated the luminescence signal that was read in the assay. Inhibition
of either of these enzymes by putative nephroprotectants would yield a
false positive in the screen.

[0170]Although compounds that inhibit luciferase are rare, a second
counterscreen was employed to eliminate these using CELLTITERGLO
(Promega). This reagent requires only viable cells (not caspase enzyme
from apoptotic cells) to generate luminescence.

[0171]Finally, compounds were tested for the ability to inhibit caspase in
an in vitro enzyme assay. Caspase inhibitors may be of interest as
general inhibitors of apoptosis that also serve to reduce nephrotoxicity,
so this screen was used to identify these compounds, but they were not
eliminated as candidate nephroprotectants.

[0172]Several compounds demonstrated a nephroprotective effect in the HK-2
assay without affecting cell viability or the luciferase readout in the
assay. All of these compounds decreased the gentamicin induced apoptosis
signal in the assay without altering the background signal. These results
demonstrated that the aforementioned compounds were directly inhibiting
aminoglycoside related nephrotoxicity and not a more general process.

[0173]The effect of these nephroprotectants on the gentamicin minimal
inhibitory concentration (MIC) in vitro is determined. Compounds that do
not raise the gentamicin MIC are considered particularly promising
candidate nephroprotectants.

Example 6

In Vitro HK-2 Cell-Based Nephrotoxicity Assay Protocol

[0174]This example describes one specific protocol for determining
nephrotoxicity of a compound according to the methods described herein.

Procedure

[0175]A human kidney proximal tubule cell line, HK-2 (ATCC #CRL-2190) is
cultured in Keratinocyte-Serum Free Medium (K-SFM) (Invitrogen 17005-042)
with 5 ng/mL of human recombinant epidermal growth factor (EGF) and 0.05
mg/mL Bovine Pituitary Extract (BPE). Culture medium containing EGF
and/or BPE is not filtered subsequent to growth factor addition, and
fresh growth factors are added to the culture medium after two weeks. The
HK-2 cells are split so that they are 80% confluent on the day they need
to be plated into 96 well plates. These cells are not allowed to become
confluent. The suggested schedule for passaging these cells is to split
them 1-2 times per week at dilutions of 1:2 or 1:3.

Day 1, Plate Cells

[0176]Remove the HK-2 cells from the plate using trypsin-EDTA. Wash the
plate with D-PBS. Add 0.5 mL trypsin, and incubate for 1 minute at room
temperature or until cells start to lift off plate. Inactivate trypsin
and harvest cells by adding prewarmed culture medium (37° C.) to
the plate. Pipet the cells with a P1000 to disassociate them, and count
the cells, adjusting them to a concentration of 1.6×105
cells/mL in culture medium. Dispense 100 μL/well (1.6×104
cells/well) into 96 well plates (tissue culture treated white plate with
clear bottom; Greiner EK-25098, E&K Scientific), and incubate the plates
at 37° C. with 5% CO2 for 3 days.

Day 4, Morning, Compound Addition

[0177]Make dilutions of experimental compounds in prewarmed assay medium
(37° C.) in dilution plates. Make at least seven 2-fold serial
dilutions starting at 500 μg/mL. Compound dilutions can be made and
stored at 4° C. for 1-4 days with a low evaporation lid and
parafilm. Warm the compound dilution plate in incubator for 10-15 minutes
prior to addition to cells. Remove the 96 well plates with HK-2 cells
from incubator, and aspirate the medium from wells. Aliquot the warmed
compounds into plates at a volume of 100 μL/well, and incubate the
plates at 37° C. with 5% CO2 overnight (˜31 hours).

[0179]The average luminescence values are calculated along with the
standard deviation of the replicates. The concentration of the
aminoglycoside tested is graphed versus the corresponding luminescence
reading. The area under the curve (AUC) is calculated for each
experimental compound and for gentamicin. The EC50 can then be calculated
for each experimental compound and gentamicin, using methods well known
in the art.

[0180]The various embodiments described above can be combined to provide
further embodiments. All of the U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign patent
applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety. Aspects of the
embodiments can be modified, if necessary to employ concepts of the
various patents, applications and publications to provide yet further
embodiments.

[0181]These and other changes can be made to the embodiments in light of
the above-detailed description. In general, in the following claims, the
terms used should not be construed to limit the claims to the specific
embodiments disclosed in the specification and the claims, but should be
construed to include all possible embodiments along with the full scope
of equivalents to which such claims are entitled. Accordingly, the claims
are not limited by the disclosure.